This proposal is directed towards our understanding of the functions and mechanisms of multiple DNA topoisomerases in eukaryotic cells. Eukaryotic DNA topoisomerase I breaks and rejoins one DNA strand at a time whereas eukaryotic DNA topoisoemrase II breaks and rejoins both DNA strands in concert. Both enzymes may be involved in controlling the complex topological state of DNA. We will investigate how these enzymes control the topological structure of DNA and how these enzymes affect various genetic processes. (1) We will study the roles of DNA topoisomerases in eukaryotic gene expression using both the Drosophila heat shock system and simian virus 40(SV40) system. (2) We will investigate the roles of DNA topoisomerases in SV40 DNA replication. We are particularly interested in the possibility that topo II may signal initiation of DNA replication by its putative gyrase activity. (3) We will try to isolate mutants of topoisomerases from Saccharomyces cerevisiae. In particular, we will use P4 knotted DNA as substrate to screen cdc mutants in vitro. (4) We will clone the human topoisomerase genes to study the protein sequence and transcriptional regulation. (5) We will study the regulation and modification of topoisomerases in cells using logical methods. (6) We will study the interactions of eukaryotic DNA topoisomerases with DNA, nucleosomes and chromosomal proteins. (7) We will investigate the possibility that DNA topoisomerases may be involved in DNA sequence rearrangement. The topoisomerase cleavage sites will be compared with the sequenced integration sites of SV40 DNA. The long-term objective of this project is to understand the functions and mechanisms of DNA topoisomerases in various genetic processes such as DNA replication, RNA transcription, recombination and nucleic acids rearrangements.